Tandem MICROFASP Method-Enabled High-Sensitivity In Vivo Cross-Linking Mass Spectrometry Analysis of Low-Cell-Number Samples without Enrichment

Cross-linking mass spectrometry (XL-MS) has emerged as an indispensable tool for dissecting protein conformations and protein-protein interactions (PPIs) within native biological systems. However, conventional in vivo XL-MS workflows suffer from significant sample loss during sample preparation, necessitating large input quantities of the starting material and fundamentally limiting their widespread applicability to mass-limited samples. Herein, we report the development of a tandem MICROFASP platform that uniquely integrates in vivo chemical cross-linking with seamless in situ sample preparation to drastically minimize sample loss throughout the entire workflow. Using disuccinimidyl suberate (DSS) as a model cross-linker, we systematically optimized all critical experimental parameters, including cross-linking reaction duration, lysis buffer formulation, and mass spectrometry data acquisition conditions. Without any pre-enrichment, we identified 517 intercross-linked peptides from just 10,000 K562 cells, a 3.5-fold increase relative to the state-of-the-art result with enrichment reported to date. Notably, 318 cross-linked peptides were identified from only 2500 living K562 cells. Structural mapping and PPI network analysis confirmed the validity of the identified cross-links, with over 97% of the cross-linked sites falling within the distance constraint of the DSS. This tandem MICROFASP method resolves the long-standing sensitivity limitation of in vivo XL-MS in low-cell-number samples, paving the way for systematic analysis of protein conformations and dynamic PPIs in clinical biopsies and the tumor microenvironment.